Diagnosis of Collagen Type III Glomerulopathy Using Picrosirius Red and PASH/Masson’s Trichrome Stain

Abstract

Canine collagen type III glomerulopathy (Col3GP) is a rare juvenile nephropathy in which irregular type III collagen fibrils and fibronectin accumulate in glomerular capillary walls and the mesangium. Necropsy findings were reviewed from 5 puppies diagnosed with Col3GP at 6 to 18 weeks of age. Histologically, with hematoxylin and eosin stain, the glomerular capillary walls and mesangium were diffusely and globally expanded by homogeneous pale eosinophilic material. Ultrastructurally, the subendothelial zone and mesangium were expanded by fibronectin and cross-banded collagen type III fibrils, diagnostic of Col3GP. Two additional stains were employed to identify the material within glomeruli as fibrillar collagen using light microscopy. In all 5 cases, the material was red with picrosirius red and birefringent under polarized light, and was blue with periodic acid-Schiff/hematoxylin/trichrome (PASH/TRI), thereby identifying it as fibrillar collagen. Based on these unique staining characteristics with picrosirius red and PASH/TRI, Col3GP may be reliably diagnosed with light microscopy alone.

Collagen type III glomerulopathy (Col3GP), also known as collagenofibrotic glomerulopathy, is a rare renal disease described in humans and several animal species.^{1,2,4–6,8–10,12–17} Individuals with Col3GP accumulate abundant type III collagen and, to a lesser extent, fibronectin and type V collagen in the mesangium and subendothelial space, adjacent to the glomerular basement membrane. This differs from the normal glomerular extracellular matrix, which contains type IV collagen, fibronectin, and other components, but lacks type III collagen.^4,5 Accumulation of fibrillar type III collagen and fibronectin in the capillary wall results in disruption of the filtration membrane, proteinuria, and progression to end-stage renal disease. Pathogenesis in humans is unknown; however, a genetic cause is presumed based on case occurrences within families.⁵

Light microscopic lesions in humans and animals with Col3GP are characterized by expansion of glomerular capillary walls and mesangium by homogeneous pale eosinophilic material. This material stains blue with Masson’s trichrome (TRI), weakly periodic acid-Schiff (PAS)-positive, argyrophilic with Jones methenamine silver impregnation, and non-congophilic.^4,5 Positive immunohistochemical labeling for type III collagen within glomerular mesangium and capillary loops is diagnostic of Col3GP; however, this assay is not offered by most diagnostic laboratories.⁴ Ultrastructural changes are diagnostic and demonstrate collagen fibrils within capillary walls and mesangium that are frayed, curved, detached from bundles, and have bands at a regular periodicity of approximately 60 nm, consistent with abnormal type III collagen.^5,14

Canine Col3GP is categorized as a juvenile nephropathy due to the onset of chronic renal failure in juvenile to young adult dogs.⁴ A simple autosomal recessive inheritance pattern in dogs is supported by a prior study; however, neither the genetic basis nor any breed predisposition has been identified.¹⁴ Previous animal cases of Col3GP include individual case reports or littermates (Table 1). The current report describes the pathologic features of Col3GP in 5 puppies. Additionally, we describe a method for diagnosing Col3GP utilizing histochemistry without the need for transmission electron microscopy (TEM) or immunohistochemistry.

Table 1.

Published Reports of Collagen Type III Glomerulopathy in Animals^a.

Reference	Species	Breed	Signalment
10	Dog	Newfoundland	2 m M, 2.5 m F, and 12 m Mb
16	Pig	Hybrid	6 m F and an adult F
17	Pig	Large White	Neonate M
12	Cat	Japanese domestic	1 y FS
6	Cynomolgus macaque	—	4.4 y M
1	Cynomolgus macaque	—	2.6 y F
8	Dog	Shiba Inu	3 y F
9	Dog	Mixed (miniature Dachshund and Chihuahua)	7 m M
14	Dog	Mixed	5 M and 9 F between 41 and 326 dc
13	Dog	Mixed	2 M and 2 F between 109 and 144 db,c
15	Dog	Drever	3 F and 1 M between 35 and 53 db

Abbreviations: M, male; F, female; FS, female spayed; d, days old; m, months old; y, years old.

^aReports listed in ascending order of publication date.

^bLittermates.

^cRelated puppies bred for the study.

Two puppies (cases 1 and 2) diagnosed with Col3GP were retrieved from the archives at the Animal Disease Diagnostic Laboratory at Purdue University. Three puppies (cases 3–5) diagnosed with Col3GP were retrieved from the archives at the Veterinary Diagnostic Laboratory at the University of Georgia, College of Veterinary Medicine. All available information including signalment, history, clinical findings, and pathologic findings were collected.

Case 1 was a 6-week-old female Pug puppy; cases 2 and 3 were 12-week-old male Pug puppies. All Pug puppies were from separate litters and submitted by different clients; relationship among the puppies was unknown. Case 4 was a 10-week-old female Bulldog (exact breed not specified). Case 5 was an 18-week-old female mixed breed (Staffordshire terrier and pit bull) puppy. Clinical history in all cases included failure to thrive with vomiting and/or poor appetite. Serum chemistry and urinalysis results were available for case 3; abnormal findings included azotemia (serum urea, 54 mg/dL; creatinine, 2.8 mg/dL), mild hypoalbuminemia, elevated serum phosphate, and a urine specific gravity of 1.022 with protein, blood, and glucose in the urine. Cause of death was not reported for case 2; all other dogs were humanely euthanized due to poor prognosis.

All 5 cases were submitted for autopsy at their respective laboratory. At autopsy, gross renal lesions were observed in cases 3, 4, and 5, including bilaterally swollen and/or pale kidneys. Other gross findings included mural thickening of the bladder wall (histologic diagnosis of histiocytic cystitis) in case 1; diffuse subcutaneous edema in case 3; cachexia and pulmonary edema in case 4; and oral, esophageal, and gastric ulcers (histologic diagnosis of disseminated soft tissue and vascular mineralization) in case 5. Case 1 was diagnosed with sepsis after culture of pooled kidney, liver, and spleen yielded Escherichia coli and Streptococcus canis.

Tissue samples obtained at autopsy were fixed in 10% neutral-buffered formalin, embedded in paraffin, and 3-μm sections were stained with hematoxylin and eosin (HE), periodic acid–Schiff and hematoxylin (PASH), and TRI. Picrosirius red and PASH/TRI combination stains were used in order to differentiate fibrillar collagens from the type IV collagen of the normal glomerular basement membrane and mesangium.^3,11 For TEM, renal tissue was retrieved from 10% buffered formalin, postfixed in 1% osmium tetroxide, serially dehydrated in graded alcohols, infiltrated with acetone (or propylene oxide) and epoxy plastic, and embedded in plastic. Sections of 1-mm thickness were cut and stained with toluidine blue to locate glomeruli before thin sections were cut at 60 nM using an ultramicrotome (Ultracut S; Reichert Technologies, Depew, NY), placed on copper slot grids coated with Formvar (Electron Microscopy Services, Hatfield, PA), and stained with uranyl acetate and lead citrate. The grids were examined in a transmission electron microscope (JEOL JEM-1210 120Kv), and multiple digital images were acquired (CAB, AMT XR41C Bottom-Mount CCD Camera).

With light microscopy of HE-stained sections, the glomerular changes in all 5 dogs were similar (Fig. 1). Glomerular capillary loops and mesangium were diffusely and globally expanded by extracellular, homogeneous, pale eosinophilic material. The mesangium was mildly to moderately hypercellular in all cases. Scattered glomeruli had segmental to global adhesions to Bowman’s capsule, which was thickened. Podocytes were variably hypertrophied and/or vacuolated. The interstitium was expanded by fibrous tissue that was most severe in older puppies. Multifocal mineralization of tubular epithelium, Bowman’s capsule, and glomerular basement membranes was most severe in older puppies.

Figure 1. Collagen type III glomerulopathy, kidney, dogs.

Case 3. Glomerular capillary loops and mesangium are expanded by pale eosinophilic, homogeneous material. Bowman’s capsule is thickened. Hematoxylin and eosin.

Transmission electron microscopy was performed on cases 1, 2, 3, and 5. The glomerular mesangium and capillary walls were markedly expanded and contained haphazardly arranged fibrils and accumulations of granular material consistent with fibronectin (Fig. 2). Capillary lumina were often narrowed by these subendothelial and intra-mesangial collagen deposits. The fibrils were 20 to 60 nm in diameter, sometimes curved, and had cross-striations on longitudinal sections with a periodicity of approximately 60 nm. There was diffuse effacement of podocyte foot processes.

Figure 2. Collagen type III glomerulopathy, kidney, dogs.

Case 2. Glomerular capillary loop. The subendothelial zone is markedly expanded by granular material and loosely arranged collagen fibrils (arrows). Podocyte foot processes are effaced (arrowhead). CL, capillary lumen; En, endothelial cell; asterisk, glomerular basement membrane. Bar = 500 nm. Inset: collagen fibrils have cross-striations, are 28 to 60 μm in diameter, and are often curved. Bar = 100 nm. Transmission electron microscopy. Uranyl acetate and lead citrate.

All 5 cases were stained with Congo red, Jones, picrosirius red, PASH, TRI, and combination PASH/TRI. The material within the glomerular capillary walls and the mesangium was not congophilic with Congo red. With Jones methenamine silver, glomerular basement membranes were strongly argyrophilic and were variably distinguishable from moderately argyrophilic material in glomerular capillary walls and the mesangium. With picrosirius red, the material expanded glomerular capillary walls and mesangium, stained red, and under polarized light, was birefringent faint yellow-red and green (Figs. 3a and b). With PASH, the material was weakly PAS-positive and was distinguished from the strongly PAS-positive basement membrane (Fig. 4a). With TRI, the material was homogeneous deep blue (Fig. 4b). With combination of PASH/TRI stain, the material was homogeneous deep blue and the glomerular basement membranes were purple (Fig. 4c). Glomeruli from a control puppy (exact age unknown) stained with PASH/TRI had purple capillary walls and mesangium (magenta from PASH plus blue from TRI produces purple; Fig. 5).

Figure 3a. Collagen type III glomerulopathy, kidney, dogs.

Case 4, picrosirius red stain. The material expanding the glomerular capillary loops and mesangium stains red. Bowman’s capsule and the interstitium are also red.

Figure 3b. Collagen type III glomerulopathy, kidney, dogs.

Case 4, picrosirius red stain under polarized light. The material expanding glomerular capillary loops and mesangium is yellow-red and green, consistent with fibrillar collagen. Bowman’s capsule and the interstitium are also yellow-red and green, consistent with fibrillar collagen.

Figure 4a. Collagen type III glomerulopathy, kidney, puppy, case 1.

Periodic acid-Schiff and hematoxylin (PASH). The material expanding glomerular capillary loops and mesangium is weakly PAS-positive. Glomerular basement membranes are strongly PAS-positive.

Figure 4b. Collagen type III glomerulopathy, kidney, puppy, case 1.

Masson’s trichrome (TRI). The material expanding glomerular capillary loops and mesangium as well as the glomerular basement membranes is dark blue, consistent with collagen.

Figure 4c. Collagen type III glomerulopathy, kidney, puppy, case 1.

PASH/TRI combination. The material expanding capillary loops and mesangium is dark blue and differentiated from the glomerular basement membranes, which are purple.

Figure 5. Collagen type III glomerulopathy, kidney, puppy, case 1.

Normal glomerulus, control puppy (exact age unknown). PASH/TRI combination. Glomerular basement membranes are purple. Dark blue material is not observed within the capillary walls and mesangium.

In human Col3GP, time of clinical presentation is divided into pediatric and adult-onset types with adult-onset being more common.⁵ In contrast, canine Col3GP is mainly reported in juvenile dogs with only one report of a dog living beyond 12 months of age.^{8–10,13–15} Despite this difference, clinical signs, light microscopy, and ultrastructural findings from the puppies of the current report were strikingly similar to those in human cases. Urinalysis and serum chemistry values were available for one dog and resembled those in human cases in which proteinuria and eventual renal failure are common findings.⁵ Although urinalysis was not available for all cases, effacement of podocyte foot processes was a feature in all puppies in which TEM was performed, and is a characteristic feature of proteinuric disorders.⁷ With light microscopy, expansion of glomerular capillary walls and the mesangium by homogeneous material that is pale eosinophilic with HE, blue with TRI, and weakly PAS-positive is described in human biopsies and was evident in all 5 puppies. Interestingly, increased mesangial cellularity is not often a feature of human cases but was a feature of all 5 puppies.^4,5,14 This may reflect the relatively acute clinical course before death or humane euthanasia in the puppies as compared to human cases. Definitive diagnosis in human cases requires TEM with evidence of irregular type III collagen fibrils in glomerular mesangium and subendothelial zone and these findings were evident in all puppies in which TEM was performed.⁵

Col3GP was diagnosed in 5 puppies using picrosirius red stain and a PASH/TRI combination to demonstrate the abnormal deposition of fibrillar collagen within glomeruli. Picrosirius red stain polarizes yellow-red and green on all types of fibrillar collagen (which is not present in the normal glomerulus) and was demonstrated in the mesangium and subendothelial spaces of these 5 dogs.¹¹ The normal glomerular basement membrane consists mainly of densely packed type IV (non-fibrillar) collagen, which is magenta with PASH; however, the accumulations of abnormal loose type III (fibrillar) collagen are only weakly PAS-positive. TRI stains all types of collagen blue.³ Therefore, the PASH/TRI combination highlights the distinction between glomerular basement membrane and abnormal collagen by staining the type III collagen blue and the type IV basement membrane collagen purple (purple as the additive effect of magenta and blue). Although this histochemical method does not definitively identify the material as type III collagen, Col3GP is the only recognized glomerulopathy in animals that has these staining characteristics with picrosirius red and PASH/TRI combination. Thus, we recommend picrosirius red and PASH/TRI combination stains to diagnose Col3GP without the need for TEM and/or immunohistochemistry.